Production of human recombinant collagen in the milk of transgenic animals

ABSTRACT

Production of human procollagen or collagen in cells which ordinarily do not produce these molecules is effected by constructing expression systems compatible with mammary glands of non-human mammals. For example, expression systems can be microinjected into fertilized oocytes and reimplanted in foster mothers and carried to term in order to obtain transgenic non-human mammals capable of producing milk containing recombinant human procollagen or collagen. Human procollagen or collagen produced in this manner can be made of a single collagen type uncontaminated by other human or non-human collagens.

This is a divisional application of a application, Ser. No. 08/183,648,filed on Jan. 18, 1994, U.S. Pat. No. 5,667,839, which is acontinuation-in-part of U.S. Ser. No. 08/011,643 filed Jan. 28 1993, nowabandoned, the contents of which are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to production of recombinant proteins,specifically collagen, in the milk of a transgenic mammal. Morespecifically, it concerns methods to prepare purified forms of usefulhuman collagen by effecting the secretion of the collagen (orprocollagen) into the milk of a transgenic mammal.

BACKGROUND ART

Collagen is a major structural protein useful in reconstructivetherapeutic procedures in humans. Collagens used for these purposes aregenerally prepared by isolating the material from tissues of farmanimals such as cows or pigs. While such isolated collagen has been usedwith some success, it is essentially a protein foreign to the treatedhuman being and immunogenic responses can be a problem. This problem hasbeen minimized by treating the animal-derived collagen with proteolyticenzymes to decrease immunogenicity.

It is clear that it would be advantageous to supply human rather thanbovine or porcine collagen for therapeutic purposes. The sources forpurified human collagen are limited and the only reliable source ishuman placenta. Human collagen can be purified from human placenta asdescribed in copending U.S. Pat. No. 5,428,022 (Collagen Corporation).The placenta contains several types of collagens, most notably types I,III, IV, and V. The process of separating and purifying one type fromthe others is imperfect and results in a predominant type with smallamounts of the other types. Production of purified collagen fromplacentas further necessitates additional processing steps to ensurethat the resulting collagen product is free from human viruses such ashepatitis and HIV. In view of this, there have been attempts to preparehuman collagen using recombinant techniques.

Expression of the human cartilage procollagen gene (Col2A1) in mouse 3T3cells been reported (Ala-Kokko, L. et al., J Biol Chem (1991)266:14175-14178). Olsen, A. S. et al. reported expression of a minigeneversion of the human proα1(I) collagen gene in mouse fibroblasts (Olsen,A. S. et al., J Biol Chem (1991) 266:1117-1121). Full-length humanproα2(V) collagen cDNA in proα2(V)-deficient hamster cells was reportedby Greenspan, D. S. et al., J Biol Chem (1989) 264:20683-20687; mousefibroblasts have also been used to express the proα1(I) chain whereinthe resulting expressed protein is complexed in the collagen triplehelix with murine proα2(I) chains, as described by Schnieke et al., ProcNatl Acad Sci USA (1987) 84:8869-8873. Transgenic mice that weremodified to contain a mutated form of the proα1(I) gene were not viableafter birth, according to a study by Stacey, A. et al. Nature (1988)322:131-136. In addition, transgenic mice have been obtained thatexpress a minigene version of the human gene for type I procollagensystemically (Khillan, J. S. et al., J Biol Chem (1991)266:23373-23379); PCT application WO92/22333. These mice are useful asmodel systems for investigating bone diseases characterized by themodified collagen produced.

The production of recombinant human collagen is made troublesome by thenecessity for a multiplicity of posttranslational enzymes which aregenerally believed to be present only in cells which natively producecollagen. At least eight such posttranslational enzymes are believed tobe needed (Prockop et al., New England J Med (1984) 311:376-386). Thishas limited attempts at recombinant production to cells which nativelyproduce this protein; this inevitably results in chimeric forms of theprotein.

In order to avoid chimeric collagens which contain partly human andpartly host mammalian cell chains in the triple helix, it might bepossible to use human cells for this production. Even in this case,however, it is not possible to obtain collagen product of a particulartype free of other collagen types. As further described below, thevariety of collagens produced and their innate similarity makeshomogeneous preparations from either native or recombinant sources whichproduce their own collagen impossible.

The present invention solves these problems by effecting the synthesisof human procollagen or collagen in cells which do not natively producethis protein, employing techniques established for the production offoreign proteins in mammalian milk, as described in the publicationscited hereinbelow. The collagen of designated types is secreted into themilk either as procollagen or collagen, depending on the construction ofthe expression systems and accompanying recombinant enzyme production.

DISCLOSURE OF THE INVENTION

The invention provides recombinant production of human collagen in aform that permits isolation of a homogeneous collagen type and can bedesigned to effect the production of commercially practical amounts ofthese proteins at a reasonable cost. The invention utilizes systemsdeveloped for the production of recombinant proteins in mammalian milkand requires utilization of these techniques not only to effect theexpression of the gene encoding the desired collagen, but also, ifrequired, expression of the gene for any required posttranslationalenzymes.

Thus, in one aspect, the invention is directed to a method for therecombinant production of human procollagen or collagen comprisingrecovering milk from the mammary glands of a nonhuman mammal. The mammalwill have been modified to contain an expression system that comprisesDNA encoding human procollagen under the control of regulatory sequencesoperable in mammary glands. The human procollagen or collagen producedis recovered from the milk by various purification techniques. Thenonhuman mammal may also be modified if necessary to contain anexpression system for the production of any needed posttranslationalenzymes in the milk protein-secreting cells of the mammary glands.

Either collagen or procollagen may be secreted depending on the presenceor absence of suitable proteases in the cell. The procollagen encoded inthe nucleotide sequence contained in the expression system will bepreceded by a nucleotide sequence encoding an appropriate signal, eitherthat natively associated with the procollagen or an alternate signalsequence workable in the targeted cells. Thus, the procollagen producedas a result of a recombinant expression will be secreted into the milk.If the host cells contain enzymes which ordinarily effect cleavage ofthe prosequences from collagen--i.e. procollagen N-protease and/orprocollagen C-protease, the procollagen will be cleaved of theprosequences as it exits the cell and collagen will be secreted into themedium. However, if these enzymes are absent from the production cell,procollagen itself will be secreted. Low levels of these proteases willresult in mixtures of collagen and procollagen. Apparently the levels ofthese enzymes vary in cells which natively produce collagen. Dependingon the tissue and the developmental stage of the subject from which thetissue originates, a greater or lesser proportion of procollagen orcollagen will be contained in the secreted materials. Thus, the milkwhich contains the collagen of the invention will contain this collagenin the form of human collagen per se, human procollagen per se or amixture of both.

While it would be possible to modify the native procollagen genes todelete the coding sequences for the prosequences, it is not desirable todo this since the pro-region, especially the C-terminal pro-region,mediates the formation of triple helixes by the collagen portion of themolecule. Thus, if the prosequences are deleted from the expressionvector, the resulting single collagen chains would be unable to form thetriple helix which characterizes the collagen molecules.

If procollagen is secreted into the milk, of course, by supplying theappropriate proteolytic enzymes, collagen will result.

In another aspect, the invention is directed to expression systemsuseful in the foregoing method which comprise a DNA sequence encodinghuman procollagen operably linked to a promoter and other regulatorysequences capable of effecting expression in mammary glands. Ifnecessary, expression systems operable in mammary glands for productionof posttranslational enzymes can also be used. The invention also isdirected to nonhuman embryonic stem (ES) cells and to nonhuman eggs,including fertilized forms, modified to contain the expression system aswell as to the nonhuman mammal implanted with the fertilized egg or witha blastula including the ES cells.

In other aspects, the invention is directed to milk containing humanprocollagen or collagen, and to homogeneous forms of human procollagenor collagen. These forms are made available by the practice of theinvention method which permits the production of only the recombinantcollagen type desired absent a background of either similar nonhumancollagen molecules, or of collagens of different types.

MODES OF CARRYING OUT THE INVENTION

Collagen is a well studied protein, and the expression of genes encodingcollagen has also been reviewed recently (Adams, S. L., Amer J RespirCell and Molec Biol (1989) 1:161-168). This review summarizes the typesof collagen known to occur and describes their common features. ThemRNAs encoding collagens of various types are translated in thecytoplasm of collagen-producing cells into procollagen subunits whichare then assembled into triple helices. The assembled procollagencontains propeptide extensions at the N and C termini that help toassemble the subunits, but do not participate in the triple helix. Theprosequences are then cleaved to obtain collagen triple helix as theprocollagen is secreted. The collagen helix itself contains nonhelicalextensions designated telopeptides. The triple helical regions containrepeating amino acid sequences with a glycine in every third positionand proline (P) or hydroxyproline (HP) often in the other positions soas to contain a sequence of "triplets" of the form --(GXY)_(n) --,wherein X or Y or both are P or HP. One of the essentialposttranslational steps is the conversion of some proline residues tohydroxyproline to ensure stability of the triple helix at bodytemperature. Other important posttranslational modifications aredisulfide exchange, hydroxylation of lysyl residues, addition ofcarbohydrate and the assembly and crosslinking of the triple helicalcollagen molecules.

According to the Adams review, thirteen genetically distinct collagentypes have been described and represent the products of at least 23genes. The most common types found in interstitial tissues are types I,III, V and VI; in cartilage, types II, IX, X and XI are found. Some ofthese types exist natively as homotriplexes; others are heterotriplexes.

The nomenclature for the various collagen types is designed to designatethe genetic origin of the collagen in question. For example, the triplehelix of type I collagen is a heterotriplex containing the products oftwo different collagen-encoding genes. This type of collagen isdesignated α₁ (I)!₂ α₂ (I); thus, type I collagen triplexes contain twochains encoded by the Col1A1 gene and one protein chain encoded by theCol1A2 gene. Type III collagen is designated α₁ (III)!₃ and is thuscomprised of three identical chains translated from the Col3A1 gene.Type II collagen is also a homopolymer designated α₁ (II)!₃ which iscomprised of translation products of the Col2A1 gene.

Since collagen-producing cells, as described above, produce severaltypes of collagen, it has, in the past, been impossible to obtain, forexample, homogeneous type I collagen free of type III collagen. Byproducing collagen in noncollagen-producing cells according to themethod of the invention, obtaining such homogeneous preparations becomespossible.

The genetic materials for use in the method of the invention encodingthe desired collagens are available. The genes encoding human types I,II, III, IV and V collagen are currently available.

Prockop, D J et al. (supra) list the following cotranslational andposttranslational modifications that occur when collagen is produced infibroblasts: cleavage of signal peptides at the N-termini of the chains,hydroxylation of the Y-position proline and lysine residues,hydroxylation of a few X-position proline residues, addition ofgalactose or galactose and then glucose to some of the hydroxylysines,addition of a mannose-rich oligosaccharide to the C propeptides,association of the C-terminal propeptides through a process directed bya structure of these domains, and finally formation of both intrachainand interchain disulfide bonds in the propeptides. After secretion ofthe procollagen, the N propeptides are cleaved by a procollagen Nproteinase and the C propeptides by a separate procollagen C proteinase.The collagen then self-assembles into fibrils, and lysyl oxidaseconverts some lysine and hydroxylysine residues to the aldehydederivatives to form cross-links with similar residues in adjacentmolecules.

It is not entirely clear whether mammary cells, since they do notendogenously produce collagen, contain the enzymes necessary for theseposttranslational events. Since the assembly into triplexes is mediatedby the sequences of the C-terminal extensions, in the event theepithelial cells of the mammary glands lack the required proteases, itis believed that the assembly into triplexes can be effectedextracellularly by providing appropriate secretion signals to theprocollagen molecule as stated above and adding suitable proteases.Alternatively, the proteases could be produced recombinantly in theepithelial mammary cells. The enzymes most likely to be needed by themammary cells in order to effect required posttranslational processingare protein disulfide isomerase and the α-subunit of prolyl hydroxylase.If these enzymes are not endogenously produced and must be providedrecombinantly, expression systems for their production may be suppliedalong with the expression systems for the collagen or procollagenitself. The gene for the α-subunit of prolyl hydroxylase has not yetbeen completely described but the gene encoding the protein disulfideisomerase has been partially sequenced as described by Tasanen, K., etal, J Biol Chem (1988) 263:16218-16224 and J Biol Chem (1992) 267:11513-11519. Genes encoding both proteins can be obtained using standardtechniques. These two enzymes function together as a tetrameric proteincomprising two subunits of prolyl hydroxylase noncovalently associatedwith two α subunits of protein disulfide isomerase. Although the twosubunits of protein disulfide isomerase are functional as a dimer, thetwo α subunits of prolyl hydroxylase must be associated with proteindisulfide isomerase in order to be active (Vuari, et al., 1992).

A well developed system for use in the invention method utilizes milkproduction in cows. This system is summarized by Krimpenfort, P. et al.in Biotechnology (1991) 9:844-847. This article describes microinjectionof fertilized bovine oocytes with genes encoding human proteins anddevelopment of the resulting embryos in surrogate mothers. The humangenes were fused to the bovine αS₁ casein regulatory elements. Thisgeneral technology was also described in PCT Application WO91/08216published Jun. 13, 1991 and assigned to GenPharm.

Additional descriptions of the production of recombinant proteins bydeveloping transgenic animals which secrete the proteins into milk arefound in European Application 264166 published Apr. 20, 1988, assignedto Integrated Genetics. This disclosure emphasizes use of whey acidprotein control systems to effect protein secretion and cites use ofthis system for the production of tPA and Hepatitis B surface antigen ingoat milk. Analogous systems for production of foreign proteins aredescribed in PCT application WO88/00239 published Jan. 14, 1988 andassigned to Pharmaceutical Proteins Limited. This application describesprocedures for obtaining suitable regulatory DNA sequences for theproducts of the mammary glands of sheep, including beta lactoglobulin,and describes the construction of transgenic sheep modified so as tosecrete foreign proteins in milk. An additional application, PCTWO88/01648, published Mar. 10, 1988 and assigned to Immunex Corporation,generally describes construction of transgenic animals which secreteforeign proteins into milk under control of the regulatory sequences ofbovine alpha lactalbumin gene. Finally, PCT application WO88/10118,published Dec. 29, 1988 and assigned to Biogen, describes constructionof transgenic mice and larger mammals for the production of variousrecombinant human proteins in milk. Other publications which describethe production of various proteins in milk include Archibold, A. L. etal. Proc Natl Acad Sci USA (1990) 87:5178-5182 which describesproduction of human α-antitrypsin in the milk of transgenic mice. Thisproduction utilized a hybrid gene constructed from the β-lactoglobulingene fused to an α1-antitrypsin minigene. Pittius, C. W. et al. ProcNatl Acad Sci USA (1988) 85:5874-5878 describe production of tissueplasminogen activator in the mammary glands of transgenic mice using themurine whey acidic protein promoter. Hennighausen, L, Protein Expressionand Purification (1990) 1:3-8 provides a review of the use of themammary gland as a bioreactor and the production of various foreignproteins in milk. This article describes the factors that affect thelevel of production and indicates recommended forms of expression systemconstruction. The disclosures of the foregoing publications areincorporated herein by reference.

Thus, techniques for construction of appropriate host vectors containingregulatory sequences effective to produce foreign proteins in mammaryglands and cause the secretion of said protein into milk are known inthe art. In addition, techniques for constructing transgenic mammalscontaining these systems, including mice as well as larger mammalianspecies such as cows, sheep and goats, are well known.

Systems for the expression of the procollagen gene in cells that producemilk protein can be constructed using methodology analogous to thatrecently described for the production of human collagenase in the lungsof transgenic mice (D'Armiento et al., Cell (1992) 71:955-961).

Genes encoding a number of procollagen types have been obtained; andgenes for additional types can be obtained similarly. The preparationand cloning of the human Col1A1 gene has been described (Barsh et al., JBiol Chem (1984) 259:14906-14913). Briefly, a human genome cosmidlibrary is packaged and used to transduce E. coli, which are plated,grown, and screened using a nucleic acid sequence specific for theCol1A1 gene. Positive colonies are located, matured in broth, and theDNA isolated. Restriction endonucleases are used to cut the DNA atselected sites. The digested DNA is examined by gel electrophoresis andDNA sequencing. A cosmid clone CG 103 isolated from a human genomiclibrary was shown to contain the entire human Col1A1 gene.

Fragments of collagen genes have been selected from cosmid libraries(Barsh et al., supra) and from bacteriophage libraries (Chu et al., JBiol Chem (1985) 260:4357-4363 for type III collagen; Chu et al., Nature(1984) 310:337-340 for type I collagen). The Col1A1 gene was obtained inthree overlapping genomic clones using the Charon 4A bacteriophagevector. The Col1A2 gene has also been obtained from five overlappingclones in Charon 4A libraries (deWet et al., J Biol Chem (1987)262:16032-16036). It has been shown that the first intron is importantin regulating the α1(1) gene expression in a tissue-specific manner intransgenic mice (Slack, J. L. et al. Mol Cell Biol (1991) 11:2066-2074).

As an alternative to using the entire gene, full-length cDNAs could beused, although the use of the entire gene has been shown to be moreeffective in transgenic animal experiments (Palmiter et al., Proc NatlAcad Sci USA (1991) 88:478-482). Such a full-length cDNA can be isolatedfrom cDNA libraries, as was done for the cDNA for the alpha-2 chain oftype I collagen (Lee et al., J Biol Chem (1988) 263:13414-13418), whichwas isolated from a lambda phage library.

To construct an expression system compatible with the epithelial cellsof mammary glands, the Col1A1 or other procollagen gene, as a DNAfragment is ligated to a similarly prepared DNA fragment containing thepromoter and any additional required regulatory sequences for amilk-specific protein expression. As described by D'Armiento et al.,when ligating a promoter to a gene, it is necessary to preserve thetranslational start site for the protein. This may be accomplished byintroducing a specific restriction endonuclease site immediatelypreceding the translation start site that is also unique for the 5' endof the chosen promoter. When these fragments are prepared using such arestriction endonuclease, the sites at the 3' end of the promoter willbe compatible with the 5' end of the Col1A1 gene. When ligation occurs,the promoter will be ligated at the correct site of the gene to encode amessenger RNA that will allow translation from the translation startsite of the procollagen gene, analogous to ligation of the heptoglobinpromoter to the human collagenase gene described by D'Armiento et al.,supra. The promoter-gene construct is ligated into a bacteriophagevector cloning system by treating the phage DNA with a restrictionendonuclease; both ends of the foreign DNA are then ligated to thevector construct for cloning the DNA.

CDNA containing the translation start site for expressed messenger RNAcan also be ligated to a promoter to prepare a functional construct forintroduction into a transgenic animal. This method was used for thehuman lactoferrin cDNA fused to the bovine alpha S1-casein gene 5' and3' untranslated regions (Krimpenfort).

It is also understood that upstream regions of the promoter may beinvolved in regulating gene expression. Specifically, it has been shownthat the extracellular matrix and hormones regulate the expression ofbovine β-casein by their influence on the upstream sequences in therelevant gene (Schmidhauser, C. et al. Proc Natl Acad Sci USA (1990)87:9118-9122). In addition, signals for termination of transcription andtranslation are also helpful in elevating levels of expression.

In order to reduce the size of the procollagen gene so that theconstruct could be cloned in bacteriophage, the gene itself could beshortened by reducing the size of the introns. This could be done forprocollagen genes that are cloned as overlapping fragments. The intronsat the junction sites of the fragments could be identified and treatedwith specific endonuclease to shorten the introns, but leave restrictionsites that are compatible for ligation. Restriction sites could bealtered by site-directed mutagenesis (D'Armiento et al., supra) togenerate restriction sites for ligation of the fragments of theprocollagen gene into a single construct. Another method ofaccomplishing the removal of introns is to prepare fusion genescontaining cDNAs to replace two or more exons within the gene.

One of the posttranslational modifying enzymes necessary for theproduction of collagen is protein disulfide isomerase, which, whencombined with the alpha subunit of prolyl hydroxylase, forms atetrameric protein isolated as prolyl hydroxylase. The gene for proteindisulfide isomerase has been obtained from a human genomic libraryproduced in a cosmid vector pcos 2EMBL (Poustka et al., Proc Natl AcadSci USA (1984) 81:4129-4133). The library was screened with cDNAfragments specific for human protein disulfide isomerase and severalclones were obtained, at least two of which contained the entire gene(Tasanen et al., J Biol Chem (1988) 263:16218-16224).

For use in the expression systems of the invention, this gene can be cutfrom the cosmid DNA with restriction endonucleases and ligated to amilk-specific protein promoter using a strategy similar to that used forthe construct of the heptoglobin-collagenase DNA.

In the event that the mammary cells are unable to provide suitableenzymes for posttranslational modification of the procollagen produced,the transgenic animals would need to be modified with expression systemsfor these enzymes. Construction of these expression systems is analogousto that described herein for procollagen gene expression. The expressionsystems for the posttranslational enzymes are provided to the transgenicanimal along with the expression systems for the desired collagenproduct.

The choice of a promoter for expression in milk would preferably be fromone of the milk-specific proteins, such as alpha S1-casein 5' and 3'regulatory sequences, which were fused to the human lactoferrin cDNA,providing a construct that used the alpha S1-casein promoter and signalsequence for the human lactoferrin gene. Another construct used toexpress a foreign protein in sheep milk consisted of the sheepbeta-lactoglobin promoter fused to human and antitrypsin gene fragments(Wright et al., Biotechnology (1991) 9:830-833). A third promoter thathas been used is the whey acid promoter, which was fused to cDNA for amodified version of human tissue plasminogen activator (Ebert et al.,Biotechnology (1991) 9:835-838) and used to prepare transgenic goats inwhose milk human tissue plasminogen activator was expressed. Thesequence of the gene is scanned for available unique restrictionendonuclease sites, which are selected so that the functional genecontaining the precise translation start site is preserved in the mRNA.

In the event that it is desirable to provide posttranslational enzymesin the mammary cells, it is believed that the most important candidatesare prolyl hydroxylase and protein disulfide isomerase. The gene for thechick alpha subunit of prolyl hydroxylase has not yet been completelyisolated, but is known to be as large as 50 kb (R. A. Berg unpublishedinformation). It is expected that the entire gene may be obtained from ahuman genomic cosmid library, as was done for the Col1A1 gene and thegene for protein disulfide isomerase. The cDNA for chick alpha subunit(Bassuk et al., Proc Natl Acad Sci USA (1989) 86:7382-7386) and humanalpha subunit (Helaakoski, T., Proc Natl Acad Sci USA (1989)86:4392-4396) have been described. Since the gene is not yet available,the cDNA for the human alpha subunit for prolyl hydroxylase can be fusedto the promoter for a milk-specific protein to produce a DNA constructfor introduction into a transgenic animal.

Using these systems, animals are obtained which secrete human collagenor procollagen into milk. The gene encoding the desired procollagenchain is coupled to suitable control sequences which function in themammary cells of mammalian species such as the regulatory sequencesassociated with the αS1 casein gene, β-lactalbumin or α-lactalbumingenes, β-lactoglobin or lactoferrin genes. Both 5' and 3' regulatorysequences can be used. The genes encoding the required posttranslationalenzymes are similarly constructed into expression systems using mammarycell-specific regulatory sequences.

The resulting expression systems are microinjected using, for example,the technique described in U.S. Pat. No. 4,873,191. The expressionsystem constructs are amplified by PCR or cloning and purified byagarose gel electrophoresis. After electroelution, the concentration isadjusted to 1-10 μg/ml and microinjected into the oocytes which areobtained from ovaries freshly removed from cows or other animals. Theoocytes are aspirated from the follicles and allowed to settle beforefertilization with thawed frozen sperm capacitated with heparin andprefractionated by Percoll gradient to isolate the motile fraction.

The fertilized oocytes are centrifuged, for example, for eight minutesat 15,000×g to visualize the pronuclei for injection and then culturedfrom the zygote to morula or blastocyst stage in oviducttissue-conditioned medium. This medium is prepared by using luminaltissues scraped from oviducts and diluted in culture medium. The zygotesmust be placed in the culture medium within two hours followingmicroinjection.

Estrous is then synchronized in the intended recipient mammals such ascattle by administering coprostanol. Estrous is produced within two daysand the embryos are transferred to the recipients 5-7 days afterestrous.

Successful transfer can be evaluated in the offspring by Southern blot.By utilizing this system to effect the expression of the Col1A1 gene,for example, the offspring can be evaluated for the presence of theCol1A1 gene by Southern hybridization using a Col1A1 gene derived probe.

Alternatively, the desired constructs can be introduced into embryonicstem cells (ES cells) and the cells cultured to ensure modification bythe transgene. The modified cells are then injected into the blastulaembryonic stage and the blastulas replaced into pseudopregnant hosts.The resulting offspring are chimeric with respect to the ES and hostcells, and nonchimeric strains which exclusively comprise the ES progenycan be obtained using conventional cross-breeding. This technique isdescribed, for example, in PCT Application WO91/10741, published Jul.25, 1991.

For production of the desired procollagen or collagen in milk,expression systems for both the procollagen gene and theposttranslational enzyme-encoding genes must be present in thetransgenic animal. There are several ways to achieve this.

First, the mammalian host may already produce the required levels ofposttranslational enzymes in the epithelial cells of the mammary glands.Alternatively, the constructs to be microinjected into eggs ortransfected into ES cells may include a cocktail of the desiredprocollagen gene expression system along with the expression systemssimilarly constructed for, for example, the prolyl hydroxylase andprotein disulfide isomerase. The successful production of collagen inthe milk can then be determined using antiprocollagen antibodies or byanalysis of the milk for levels of hydroxyproline, a unique amino acidfound in collagen as a result of the activity of prolyl hydroxylase.

In another alternative, the expression systems for the procollagen geneand the expression systems for any needed posttranslationalenzyme-encoding genes may be injected into different batches offertilized eggs or transfected into different batches of ES cells andused separately as described above to develop transgenic animals capableof expressing the procollagen or collagen genes and theposttranslational enzyme-encoding genes, respectively. These transgenicanimals can then be crossbred and the offspring evaluated for theability to express both such systems. At least some of the offspring ofsuch transgenic animals will be capable of producing both the collagenproduct and the posttranslational enzyme product.

In still another approach, fertilized eggs or ES cells may be preparedfrom transgenic animals already modified to have the capacity to expressone or the other of the procollagen genes or the posttranslationalenzyme-encoding genes. These eggs can then be microinjected or the EScells transfected with the expression system for the proteins lacking inthe transgenic animal to develop into a transgenic animal containingexpression systems for all of the required components.

Similarly, transgenic animals already modified with respect to onedesired gene may be used as sources for the blastulas into whichmodified ES cells are implanted. Again, chimeric animals will resultwhich can be used in cross-breeding to obtain offspring having genes forall of the desired proteins.

It may be noted that the expression systems for both of the particularposttranslational enzymes described above, if needed, must be providedessentially simultaneously since the enzymes function together as atetrameric protein; as described above, the two α subunits of prolylhydroxylase must be associated with protein disulfide isomerase in orderto be active.

When suitable transgenic mammals have been obtained by any of theforegoing methods, the procollagen or collagen is secreted into themilk. The procollagen or collagen product of the transgenic mammal willbe determined by the nature of the procollagen gene in the expressionsystem provided. For homotriplexes, only a single gene is inserted. Forproduction of heterotriplexes, such as typical human collagen type I,either both the Col1A1 and Col1A2 genes are utilized in the originalmicroinjection, or mammals transgenic for human Col1A1 are crossbredwith mammals transgenic for human Col1A2. The type III collagen geneCol3A1 can be used to prepare a transgenic animal and may be simplerbecause only one collagen polypeptide chain is required.

For the procollagen genes provided in the expression systems,procollagen is secreted into the milk if the required proteases forconversion to collagen are absent. To the extent that these proteaseenzymes are absent from the secreting epithelial cells and are notprovided for by recombinant systems, procollagen is secreted into themilk and can be recovered in a manner analogous to procedures that wouldbe used for collagen per se. The procollagen can also be convertedbefore or after purification using specific proteases to cleave theprosequences as is known in the art. On the other hand, if the proteasesare natively present intracellularly or are provided by recombinantsystems, collagen will be secreted directly. Depending on the levels ofthese enzymes, mixtures of procollagen and collagen may be obtained inthe milk which can, if desired, be converted by treatment of the milkwith proteases to convert all of the relevant molecules to collagen perse.

As described above, previous preparations of human collagen of a giventype are always contaminated by the presence of alternative typecollagens in view of the similarity of these materials and in view ofthe capacity of native or other recombinant cells previously used toproduce collagens encoded by their own genomes. By use of the method ofthe invention, it is possible to obtain collagen or procollagen of agiven type free from coexpressed collagens or procollagens ofalternative types.

Purification of collagen or procollagen from milk is accomplished usingtheir characteristic solubility and chemical properties. For example,milk may be acidified, causing milk-specific proteins such as casein toprecipitate and collagen or procollagen to remain in solution. Thecollagen or procollagen may be precipitated from acid solutions by theaddition of salt, alcohol, or propylene glycol. (Miller, E. J. andRhodes, R. K., Methods in Enzymology (1982) 82:33-64); Sage, H. andBernstein, P., ibid., 96-127.)

I claim:
 1. A method of preparing a single type of trimeric humanprocollagen, collagen, or a mixture of such procollagen and collagen,which method comprises the step of:recovering said procollagen,collagen, or mixture from milk of a transgenic mammal comprising anexpression system comprising a coding nucleotide sequence encoding ahuman procollagen polypeptide chain operably linked to a controlnucleotide sequence that effects expression specifically in milkprotein-secreting epithelial cells of a mammary gland in said mammal,and said cells express said coding nucleotide sequence to produce saidpolypeptide chain, and secrete said procollagen, collagen, or mixturecomprising said chain in said milk without extracellular aggregation ofsaid procollagen, collagen or mixture which would prevent excretion fromsaid mammary gland.
 2. The method of claim 1, wherein said controlnucleotide sequence comprises a bovine alpha S1-casein promoter DNAsequence.
 3. The method of claim 2, wherein said single type of humanprocollagen is human Type I procollagen and said single type of humancollagen is human Type I collagen.
 4. A transgenic nonhuman mammal whosesomatic and germ cells contain an expression system comprising a codingnucleotide sequence encoding a human procollagen polypeptide chainoperably linked to a control nucleotide sequence that effects expressionspecifically in milk protein-secreting epithelial cells of a mammarygland in said mammal, and said cells express said coding nucleotidesequence to produce said polypeptide chain, and secrete a single type ofhuman procollagen, collagen, or a mixture thereof into the milk of saidmammal, wherein said milk comprises trimeric collagen, procollagen, or amixture thereof, in recoverable amounts without extracellularaggregation which would prevent excretion from said mammary gland. 5.The transgenic nonhuman mammal of claim 4, wherein said single type ofhuman procollagen is human Type I procollagen and said single type ofhuman collagen is human Type I collagen.
 6. The transgenic nonhumanmammal of claim 4, wherein said promoter is a bovine alpha S1-caseinpromoter.